Process for the preparation of dipeptides with c-terminal non-proteinogenous amino acids

ABSTRACT

Process for producing dipeptides from non-proteinogenic amino acids with terminal carbon atoms.

DESCRIPTION

The present invention relates to a process for preparing dipeptidesderived from non-proteinogenous amino acids.

The specific synthesis of peptides which contain non-proteinogenousamino acids entails stepwise synthesis of the desired peptides employingthe unnatural amino acids and using conventional protective groups andactivation methods.

In this strategy, the unnatural amino acids employed are obtained eitherby non-enantioselective synthesis and subsequent racemate resolution orby enantioselective synthesis using optically active auxiliaries.

Difficulties in forming the peptide linkage can occur especially whenthe α position of the unnatural amino acid is substituted by twosterically demanding radicals (Helv. Chim. Acta 69, (1986) 1153, J.Amer. Chem. Soc. 103 (1981) 6127).

It has now been found that certain dipeptides can be prepared in astraightforward manner.

The invention relates to a process for preparing dipeptides withC-terminal non-proteinogenous amino acids, of the formula I ##STR1## inwhich R¹ is a C₁ -C₈ -alkyl, phenyl or benzyl group, R² denotes a C₁ -C₈-alkyl group which can be interrupted by --O--, --S--, --CO-- or--CO--O--, or a phenyl or benzyl group, R³ is a hydrogen atom or a C₁-C₈ -alkyl group or represents together with R² the radicals --(CH₂)₃--, --(CH₂)₄ -- or --CH₂ --CH═CH--CH₂ --, and R⁴ denotes a methyl,isopropyl, isobutyl, 2-butyl, t-butyl or benzyl radical, R⁵ is a methylor ethyl group, which comprises hydrolyzing a compound of the formula II##STR2## in which R¹ to R⁵ have the stated meaning.

The hydrolysis of the compounds II to give the final products takesplace especially well in dilute aqueous mineral acids in the presence oforganic cosolvents such as methanol, ethanol, tetrahydrofuran,acetonitrile or dioxane. The mineral acids which are preferably used areHCl, HBr and HI. The reaction is normally carried out at from 0° to 40°C. The reaction takes 15 min to 6 h.

The compounds II can be obtained by selective partial acid-catalyzedcleavage of the compounds III ##STR3## The selective partialacid-catalyzed cleavage is carried out by reacting the compounds IIIwith one equivalent of acid in an inert aprotic solvent. Acids whichshould be particularly mentioned are HCl, HBr and HI, and Lewis acidssuch as trialkylsilyl halides or dialkoxyboron halides. Suitablesolvents are diethyl ether, dibutyl ether, tetrahydrofuran, dioxane,dichloromethane, tetrachloromethane, toluene, cyclohexane and ethyleneglycol dimethyl ether. The reaction is usually carried out at from 0° to40° C. The reaction takes 15 min to 6 h.

The compounds II in which R³ is hydrogen can be converted bybase-induced alkylation with alkyl halides or alkyl sulfates into thecompounds II in which R³ is a C₁ -C₈ -alkyl group.

The compounds of the formula II in which R³ is a hydrogen atom and R⁵ isan ethyl group can be prepared especially well from the compounds of theformula IV ##STR4## by demethylation with a trialkylsilyl iodide with1-4 carbon atoms in the alkyl radicals. The reaction is expedientlycarried out at from 0° to -20° C. in an anhydrous halohydrocarbon assolvent and under an inert gas. The trialkylsilyl iodides which arepreferably used are those of the formula ##STR5## in which R⁶ -R⁸ areC₁₋₄ alkyl radicals.

Because some of the compounds I in the form of the free bases readilyform the corresponding diketopiperazines by ring closure, the salts of Iproduced in the hydrolysis are preferably converted by reaction with theacylating agents customary in peptide chemistry, such asbenzyloxycarbonyl chloride, 9-fluorenylmethoxycarbonyl chloride ordi-tert-butyl carbonate into the N-protected derivatives (cf.Houben-Weyl, Methoden der organischen Chemie, Vol. 15/1, G. ThiemeVerlag, Stuttgart 1974) or reacted with activated amino-acid derivatives(cf. Houben-Weyl, Methoden der organischen Chemie, Vol. 15/2, G. ThiemeVerlag, Stuttgart 1974) to give tripeptide derivatives.

The compounds I have two asymmetric carbon atoms which can have the (R)and (S) configuration and whose configuration can be identical ordifferent.

The starting materials III required for the synthesis of the dipeptidesare known, cf. Pure Appl. Chem. 55, 1799 (1983) Angew. Chem. 99, 137(1987) and earlier publications in this series.

The compounds IV in which R⁵ represents an ethyl group can be preparedparticularly straightforwardly by reacting a compound of the formula V##STR6## with a mono- or dialkylamine in the presence of a trialkylborate or of another mild Lewis acid to give a compound of the formulaVI ##STR7## in which R⁶ represents a hydrogen atom or a C₁ -C₆ -alkylgroup and R⁷ represents a C₁ -C₆ -alkyl group, removing the amineradical by hydrolysis and subsequently ethylating the hydrolysis productin the 6 position with triethyloxonium tetrafluoborate. The resultingmixed bislactim ethers of the formula VII ##STR8## (VII=IV, R¹ =R² =H,R⁵ =C₂ H₅) can subsequently be converted by single or doubleC-alkylation of their lithium salts into the intermediates IV.

Conversion of V into VI is preferably carried out by reacting V with amethanolic solution of about 1.5 equivalents of mono- or dialkylamine inthe presence of catalytic quantities of a Lewis acid at from 20° to 60°C. The compounds VI are purified by distillation.

The conversion of VI into VII takes place in two steps. In the firststep, the amino group is eliminated by hydrolysis. For this, VI isstirred in water at pH 6 to 9 at 50° to 100° C. The mixture is thenextracted with a solvent such as halohydrocarbon or ethyl acetate, andthe resulting monolactim ether is alkylated with triethyloxoniumtetraborate (sic). The alkylation is carried out in a chlorohydrocarbonat room temperature and usually takes from 1 to 3 days. The alkylationis followed by neutralization of the solution and extraction of thecompound VII.

The process according to the invention for preparing theα-alkyl-branched dipeptides is distinguished by the fact that theunnatural amino acid is built up on a lactim ether derivative, i.e. amasked dipeptide derivative, which can be obtained without difficulty.This chiral "auxiliary" II becomes part of the desired dipeptide. Theinvention makes it possible to cleave only one ether linkage in thecompounds III and IV. Thus, surprisingly, the monolactim ether II is thevery predominant product from III with one equivalent of acid. Anyregional isomers which are produced are removed by chromatography ordistillation. Compounds of the formula II can be obtained in aparticularly straight-forward manner from IV by selective demethylationwith trialkylsilyl iodide. I is obtained from II by selective hydrolysiswith aqueous acid.

Compounds of the formula I and N-protected derivatives which can beprepared therefrom in a straightforward manner by reaction with theconventional acylating agents such as benzyloxycarbonyl chloride,9-fluorenylmethoxycarbonyl chloride or di-tert-butyl dicarbonate ((BOC)₂O) are used as building blocks for synthesizing biologically activepeptides. The process is suitable for preparing building blocks forpeptides or peptide analogues which inhibit the enzyme renin, are activeon oral intake and thus can be used for treating high blood pressure.

EXAMPLES

a. BOC-L-Val-R-α MePhe-OMe

1.44 g of(2S,5R)-2,5-dihydro-2-isopropyl-3,6-dimethoxy-5-benzyl-5-methylpyrazinein 30 ml of diethyl ether were treated at room temperature with 25 ml of0.2 N HCl in diethyl ether. After 2 h, the mixture was evaporated underreduced pressure. Distillation of the residue yielded 0.94 g (69%) ofcrude(2S,5R)-2,5-dihydro-2-isopropyl-6-methoxy-5-benzyl-5-methyl-3-pyrazinone,boiling point 170° C./0.005 torr.

50 ml of 0.1 N HCl were added to a solution of 0.35 g of the pyrazinoneobtained above in 50 ml of acetonitrile. Reaction was then carried outwith stirring at room temperature for 24 h. Working up initiallyentailed removal of unreacted pyrazinone by extraction with 3×30 ml ofdichloromethane. The aqueous phase was adjusted to pH 9 with ammonia andextracted with dichloromethane (3×10 ml). The organic extract was driedover magnesium sulfate and evaporated to dryness under reduced pressure.0.3 g of crude H-Val-R-αMePhe-OMe was obtained and was immediatelyconverted by the conventional process with di-tert-butyldicarbonate/triethylamine in dichloromethane into the BOC compound.Purification by chromatography on silica gel (elution with 1:3ether/petroleum ether) yielded 0.35 g (90%) of BOC-L-Val-R-αMePhe-OMe,melting point 57° C.

2. BOC-L-Val-R-αMePro-OMe

0.45 g of(2S,5R)-2,5-dihydro-2-isopropyl-5-methyl-6-methoxypyrrolidino-3-pyrazinonein 20 ml of tetrahydrofuran were mixed with 30 ml of 3 N HCl at roomtemperature. Evaporation to dryness and reaction with di-tert-butyldicarbonate in triethylamine yielded after purification bychromatography on silica gel (elution with 1:1 ether/petroleum ether)0.43 g of BOC-L-Val-R-αMePro-OMe, melting point 92°-95° C.

The following were obtained analogously:

3. Methyl BOC-L-R-αMe-pipecolate; melting point 81°-85° C.;

4. Methyl BOC-L-Val-R-αMe-baikiate; melting point 31°-35° C.

5. BOC-L-Val-(D)-N-methyl-Me-Phe-OEt

a) 0.3 g of(2R,5S)-2-benzyl-2,5-dihydro-3-ethoxy-5-isopropyl-6-methoxy-2-methylpyrazinewas dissolved in 15 ml of absolute dichloromethane and cooled underargon to -10° C. Then 25 mg of iodine and 0.16 ml of trimethylsilyliodide were added. After 3 h, the solution was allowed to warm to roomtemperature and was stirred at 22° C. for 14 h. 5 ml of ethanol and 5 mlof diethylamine were added and the mixture was then evaporated underreduced pressure and the residue was chromatographed on silica gel(ether, Rf=0.44). 0.29 g (91%) of(2R,5S)-2-benzyl-3-ethoxy-5-isopropyl-1,2,5,6-tetrahydro-2-methyl-6-pyrazinone,melting point 125° C., was obtained.

b) 0.25 g of the product obtained in a) was dissolved in 10 ml oftetrahydrofuran and, after addition of 7 mg of sodium hydride, stirredat room temperature for 1.5 h. After addition of 0.5 ml of dimethylsulfate, the mixture was stirred for a further 10 h. Then 2 ml ofdimethylamine and 4 ml of ethanol were added and the solvent wasstripped off. Chromatography on silica gel (1:1 diethyl ether/petroleumether, Rf=0.14) yielded 0.25 g (96%) of(2R,5S)-2-benzyl-3-ethoxy-5-isopropyl-1,2,5,6-tetrahydro-2-methyl-6-pyrazinone.

c) 0.2 g of the product obtained in b) was dissolved in 10 mlacetonitrile, and 2.7 ml of 1 N hydrochloric acid were added. Themixture was left to stand at room temperature for 5 h and thenconcentrated under reduced pressure. The residue was dissolved in 1.2 mlof triethylamine and 10 ml of dichloromethane, and 0.3 g of BOCanhydride was added. The mixture was stirred at room temperature for 12h and then concentrated under reduced pressure. The residue was . . .(sic) by chromatography on silica gel (3:1 petroleum ether/diethylether, Rf=0.11). 0.26 g (85%) of product was obtained.

C₂₃ H₃₆ N₂ O₅ (420.55); Calc.: C=65.69, H=8.63. Found: C=65.51, H=8.60.

6. (L)-N-BOC-Val-(D)-Me-Phe-OEt

The example was repeated without process step b). The final product wasobtained in 83% yield, melting point 102° C.

We claim:
 1. Process for preparing dipeptides with C-terminalnon-proteinogenous amino acids, of the formula I ##STR9## in which R¹ isa C₁ -C₈ -alkyl, phenyl or benzyl group, R² denotes a C₁ -C₈ -alkylgroup which can be interrupted by --O--, --S--, --CO-- or --CO--O--, ora phenyl or benzyl group, R³ is a hydrogen atom or a C₁ -C₈ -alkyl groupor represents together with R² the radicals --(CH₂)₃ --, --(CH₂)₄ -- or--CH₂ --CH═CH--CH₂ --, and R⁴ denotes a methyl, isopropyl, isobutyl,2-butyl, t-butyl or benzyl radical, R⁵ represents a methyl or ethylgroup, characterized in that a compound of the formula II ##STR10## inwhich R¹ to R⁵ have the stated meaning, is hydrolyzed.
 2. Processaccording to claim 1, characterized in that the compounds of the formulaII in which R³ denotes hydrogen or a C₁ -C₈ -alkyl radical are obtainedby selective partial cleavage of compounds of the formula III ##STR11##in which R¹, R², R⁴ and R⁵ have the meaning specified in claim 1, andsubsequent N-alkylation where appropriate.
 3. Process according to claim1, characterized in that the compounds of the formula II (R³ =H) areobtained by demethylation of compounds of the formula IV ##STR12## inwhich R¹, R² and R⁴ have the meaning specified in claim 1, with atrialkylsilyl iodide.